This proposal describes a project to learn how cells regulate the assembly of a complex, microtubule-containing organelle, the flagellum. Although the regulation of assembly of singlet microtubules, such as those in the mitotic spindle, has been intensively studied, almost nothing is known about how cells regulate the assembly of the more stable doublet microtubules of cilia and flagella, or the triplet microtubules of the basal bodies and centrioles. We will use mutants which do not properly control their flagellar length as an entry point to dissect the regulation of flagellar assembly. Mutation in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutations in four unlinked genes have been shown to cause cells to lose flagellar length control. The mutants grow flagella of up to four times normal length. A number of these mutants, paradoxically, show severe defects in the regrowth of flagella after amputation. During the preceding project period we cloned three of these genes, LF2, LF3 and LF4. This proposal describes experiments to use a broad range of techniques to characterize the LF genes, their protein products and the proteins with which they interact to control flagellar length. The LF1 gene will be cloned, and the gens and full length cDNAs for all four LF genes will be sequenced. The protein products will be localized in wild-type and mutant cells. Powerful suppressor screens will be used to isolate new mutant alleles of each of the genes, with a particular goal of identifying missense mutations for interactive suppressor screens and for structure/function studies Both molecular and genetic techniques will be used to identify and characterize proteins which interact with the LF gene products in the regulation of flagellar length. Given the high degree of conservation of both the structure and protein composition of cilia and flagella, this project should provide useful insights into the assembly of these organelles in humans.